Maternal Lipid Metabolism Directs Fetal Liver Programming … · 2019-10-30 · Fasting induces...

Article

Maternal Lipid Metabolism
Directs Fetal LiverProgramming following Nutrient Stress
Graphical Abstract

Highlights

d Maternal fasting rescues deficits in fetal pyruvatemetabolism

d Maternal lipid metabolism drives the fetal response to fasting

d Deficits in maternal lipid catabolism potentiate the fetal

response to fasting

d The fetal response to fasting is Ppara dependent

Bowman et al., 2019, Cell Reports 29, 1299–1310October 29, 2019 ª 2019 The Author(s).https://doi.org/10.1016/j.celrep.2019.09.053

Authors

Caitlyn E. Bowman,

Ebru S. Selen Alpergin, Kyle Cavagnini,

Danielle M. Smith, Susanna Scafidi,

Michael J. Wolfgang

[email protected]

In Brief

Pregnancy is a time of incredible

metabolic demand that necessitates

metabolic communication between

mother and fetus. Using multiple mouse

models with impaired carbohydrate and/

or fatty acid metabolism, Bowman et al.

show the maternal requirements during

nutrient deprivation that drive metabolic

and transcriptional programming in the

fetus.

mailto:[email protected]

https://doi.org/10.1016/j.celrep.2019.09.053

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Cell Reports

Article

Maternal Lipid Metabolism Directs Fetal LiverProgramming following Nutrient StressCaitlyn E. Bowman,1 Ebru S. Selen Alpergin,1 Kyle Cavagnini,1 Danielle M. Smith,1 Susanna Scafidi,3

and Michael J. Wolfgang1,2,4,*1Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA2Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA3Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA4Lead Contact*Correspondence: [email protected]


SUMMARY

The extreme metabolic demands of pregnancyrequire coordinated metabolic adaptations betweenmother and fetus to balance fetal growth andmaternal health with nutrient availability. To deter-mine maternal and fetal contributions to metabolicflexibility during gestation, pregnant mice with ge-netic impairments in mitochondrial carbohydrateand/or lipid metabolism were subjected to nutrientdeprivation. The maternal fasting response initiatesa fetal liver transcriptional program marked by upre-gulation of lipid- and peroxisome proliferator-acti-vated receptor alpha (Ppara)-regulated genes.Impaired maternal lipid metabolism alters circulatinglipid metabolite concentrations and enhances thefetal response to fasting, which is largely dependenton fetal Ppara. Maternal fasting also improves meta-bolic deficits in fetal carbohydrate metabolism byincreasing the availability of alternative substrates.Impairment of both carbohydrate and lipid meta-bolism in pregnant dams further exacerbates thefetal liver transcriptional response to nutrient depri-vation. Together, these data demonstrate a regulato-ry role formitochondrial macronutrientmetabolism inmediating maternal-fetal metabolic communication,particularly when nutrients are limited.

INTRODUCTION

Pregnancy exerts an extreme metabolic demand that necessi-

tates coordinated adaptations between mother and fetus to

ensure that the energetic and biosynthetic requirements of the

rapidly growing fetus are met without compromising maternal

health and fecundity. Genetic and environmental perturbations

that disrupt this intimate communication can lead tomaladaptive

metabolic responses and serious adverse consequences for

both mother and fetus. While hormonal cues from both mother

and conceptus can affect nutrient mobilization, the metabolic

demands of the growing fetus can also directly modify maternal

Cell RepThis is an open access article under the CC BY-N

metabolism and behavior (Freinkel, 1980; Yamashita et al.,

2000). The metabolic demands of pregnancy are incredibly

high compared to non-pregnant states and nutrient withdrawal

is especially challenging, particularly during late gestation

when the conceptus is large enough to challenge maternal en-

ergy reserves. Fasting in late gestation during religious observa-

tion or illness results in earlier and more dramatic shifts to

alternative oxidative substrates such as circulating ketone

bodies, one hallmark of the accelerated starvation response

characteristic of pregnancy (Boden, 1996; Freinkel, 1980).

Even though oxygen tension in utero is low relative to atmo-

spheric levels, fetal oxidative metabolism is critical for fetal

growth and development as evidenced by the host of physiolog-

ical adaptations in place to ensure the adequate transport

of glucose and oxygen to the conceptus (Blackburn, 2007).

Whereas anaerobic metabolism dominates in early develop-

ment, once placental exchange matures and fetal mitochondrial

biogenesis accelerates (Ebert and Baker, 2013), the fetus is

poised to utilize oxidative metabolism for energy production.

The shift from anaerobic to aerobic metabolism continues to

accelerate in late gestation to prepare the fetus for the oxidative

environment of postnatal life.

Previously, we developed models of impaired mitochondrial

carbohydrate and fatty acidmetabolism that exhibit maladaptive

responses to nutrient deprivation. The selective loss of fatty acid

oxidation from the liver by the hepatocyte-specific deletion of

carnitine palmitoyltransferase 2 results in mice (Cpt2L�/�) with

elevated circulating lipids that cannot generate ketone bodies

during a fast and that, remarkably, maintain fasting euglycemia

via extrahepatic gluconeogenesis (Lee et al., 2016b, 2017). Addi-

tionally, we developed amousemodel of mitochondrial pyruvate

carrier (MPC) deficiency by generating a hypomorphic knockin

(KI) allele of Mpc1 that results in early perinatal lethality (Bowman

et al., 2016). While the complete loss of Mpc1 is not compatible

with embryonic development, a surprisingly small quantity of the

MPC is sufficient for mammalian development due to an unex-

pected metabolic flexibility of the growing fetus. Among the

alternative substrates that contributed to meeting metabolic de-

mand during late gestation were amino acids and lipids, and de-

fects in embryonic development could be rescued by feeding

dams a ketogenic diet (Sussman et al., 2013; Vanderperre

et al., 2016). Given that the ketogenic diet mimics fasting, we

sought to determine the metabolic flexibility required during

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Figure 1. Maternal Fasting Ameliorates Metabolic Derangements in Mpc1-Deficient Fetuses

(A) Immunoblot for Mpc1 and Mpc2 in maternal liver and fetal liver, 17.5 dpc, fed or 24 h fasted.

(B) Maternal body weights and changes in weight from 16.5 to 17.5 dpc.

(C) Late-gestation (e17.5) fetal body weights from fed or fasted Mpc1KI/+ dams.

(legend continued on next page)

1300 Cell Reports 29, 1299–1310, October 29, 2019

maternal nutrient deprivation to support fetal growth and devel-

opment by utilizing models with genetically tractable metabolic

perturbations in key regulatory nodes in mother and fetus.

Here, we have shown that defects in fetal pyruvatemetabolism

can be rescued by fasting-induced changes to the maternal

metabolic milieu. The loss of hepatic fatty acid oxidation from

the maternal but not fetal compartment affects fetal metabolic

and transcriptional programming. Additionally, the lipid-medi-

ated control of fetal transcriptional programming is phenocopied

in an orthogonal model of deficient fatty acid catabolism, perox-

isome proliferator-activated receptor alpha (Ppara) knockout

(KO) dams, that also revealed a dependence on fetal Ppara

signaling. Finally, the combined loss of mitochondrial pyruvate

metabolism and hepatic fatty acid oxidation further exaggerated

the fetal metabolic and transcriptional response to maternal

nutrient stress. These data show the critical role of the maternal

metabolic environment in dictating fetal metabolic and transcrip-

tional programming.

RESULTS

Maternal Response to Nutrient Deprivation withImpaired Mitochondrial Pyruvate TransportThe rapidly growing fetus requires a continuous delivery of

glucose and oxygen that places an incredible demand on

maternal metabolism. The metabolism of glucose and oxygen

converge at the transport of pyruvate into mitochondria via the

MPC. We previously showed that late-gestation (e17.5) fetuses

with a hypomorphic KI allele of Mpc1 exhibited impaired growth

and lactic acidosis (Bowman et al., 2016). To understand the role

of impaired pyruvate metabolism on the maternal and fetal

response to ametabolic challenge during late gestation, we sub-

jected both wild-type (WT) andMpc1 KI heterozygous (Mpc1KI/+)

pregnant dams to a 24-h period of food deprivation from 16.5 to

17.5 days post coitum (dpc). Relative to WT controls, Mpc1KI/+

dams exhibited a 50% reduction andMpc1KI/KI fetuses exhibited

undetectable Mpc1 and Mpc2 protein expression in liver in both

the fed and fasted states (Figure 1A). The 24-h fasting paradigm

induced an 8% loss in maternal body weight in both genotypes

(Figure 1B) and was sufficient to stunt fetal growth of WT and

Mpc1KI/+ littermates while Mpc1KI/KI fetal body weights were

not further perturbed by maternal fasting (Figure 1C). Fasting

induced a marked upregulation of circulating b-hydroxybutyrate

in both WT and Mpc1KI/+ dams (Figure 1D), consistent with the

accelerated fasting response observed during pregnancy (Frein-

kel, 1980; Metzger et al., 1982). Serum non-esterified free fatty

acid (NEFA) levels were also upregulated by fasting, and

Mpc1KI/+ dams exhibited a slight decrease in circulating NEFA

(D) Maternal blood glucose, serum b-hydroxybutyrate, and serum non-esterifie

glyceride content normalized to protein content (mean ± SEM, n = 5–6).

(E) Steady-statemetabolite abundances from e17.5 fetal liver, determined by 1H-N

n = 6).

(F) Lactate concentrations from fetal brain and heart measured enzymatically (m

(G) Intracellular lactate concentrations from Mpc1 deletion (D/D) mouse embry

ybutyrate (bHB) for 24 h, determined by 1H-NMR (mean ± SEM, n = 3).

(H) Triglyceride content of placenta and liver at e17.5, determined enzymatically

Statistically significant differences (p < 0.05) for pairwise comparisons after ANO

concentrations and liver triglyceride content relative to WT

dams (Figure 1D). Together, these data suggest that Mpc1KI/+

dams rely more on the utilization of fatty acids as an alternative

energy source upon impaired mitochondrial pyruvate meta-

bolism, as others have implicated in non-pregnant rodent

models of MPC deficiency (Gray et al., 2015; Li et al., 2016;

McCommis et al., 2015; Zou et al., 2018).

Maternal Fasting Rescues Metabolic Dysfunction inMpc1-Deficient FetusesTo better characterize the nutrient allocation between the

maternal and fetal compartments, we compared steady-state

maternal serummetabolites andmaternal liver and fetal liver me-

tabolites by 1H-NMR metabolomics. Consistent with the

decrease in blood glucose and increase in serum b-hydroxybu-

tyrate upon fasting, concentrations of these metabolites in

maternal liver were similarly regulated (Table S1). As expected,

Mpc1KI/KI fetal livers exhibited elevated lactate relative to WT lit-

termates. Interestingly, maternal fasting rescued the lactic

acidosis in Mpc1KI/KI fetal liver by reducing lactate concentra-

tions to WT levels (Figure 1E). Mpc1KI/KI fetal brain lactate con-

centrations were also ameliorated by maternal fasting, while

lactate levels in heart tissuewere not regulated bymaternal nutri-

tional status (Figure 1F). Fetal liver aspartate was also high in

Mpc1KI/KI fetuses and was suppressed by fasting similar to liver

lactate concentrations (Figure 1E). Fasting induced a greater

than 5-fold increase in b-hydroxybutyrate (bHB) levels in fetal

liver, and Mpc1KI/KI fetal livers had higher concentrations

than WT littermates (Figure 1E). To test whether bHB may

have contributed to the fasting-induced suppression of lactic

acidosis, WT and Mpc1 deletion (Mpc1D/D) mouse embryonic fi-

broblasts (MEFs) were cultured in the presence or absence of

1 mM bHB for 24 h, and steady-state intracellular lactate con-

centrations were measured. Consistent with its role as a fast-

ing-induced alternative oxidative substrate, bHB treatment was

sufficient to rescue lactate accumulation in cells lacking the

MPC (Figure 1G). These data are consistent with the finding

that a maternal ketogenic diet can rescue fetal lactic acidosis

and developmental delay in another model of impaired MPC

function (Vanderperre et al., 2016). Together, these data show

that maternal fasting can ameliorate fetal defects in mitochon-

drial pyruvate metabolism.

Fasting induces adipose triglyceride mobilization and subse-

quent hepatic ketone generation, and both of these processes

are increased during late gestation. To begin to understand the

role of enhanced lipid mobilization during pregnancy (Elliott,

1975; Knopp et al., 1970), we measured several indicators of

fetal lipid accrual during maternal fasting. First, we observed

d fatty acid (NEFA) concentrations (mean ± SEM, n = 5–6). Maternal liver tri-

MR. Normalized to protein content and shown relative toWT fed (mean ±SEM,

ean ± SEM, n = 6).

onic fibroblasts (MEFs) under standard culture conditions ± 1 mM b-hydrox-

(mean ± SEM, n = 5–8). Data from WT fetuses of WT dams are shaded green.

VA indicated by letters.

Cell Reports 29, 1299–1310, October 29, 2019 1301

Figure 2. Maternal Lipid Metabolism Pro-

vides Substrates for Fetal Metabolism

(A) Blood glucose, serum b-hydroxybutyrate,

NEFA, and triglyceride levels from fed and 24-h-

fasted liver-specific Cpt2 KO pregnant dams

(Cpt2L�/�) and Cpt2f/f (WT) controls at 17.5 dpc

(mean ± SEM, n = 6–8).

(B) Fetal body weights of control and Cpt2L�/�

progeny from control and Cpt2L�/� pregnancies

(mean ± SEM, sample size indicated).

(C) Fetal liver b-hydroxybutyrate concentrations

determined by 1H-NMR (mean ± SEM, n = 6).

(D) Triglyceride content of placenta and liver at

e17.5, determined enzymatically (mean ± SEM,

n = 5–6).

(E) Maternal blood acylcarnitine abundances at

17.5 dpc determined from dried blood spots after

extraction, butylation, and tandem mass spec-

trometry (mean ± SEM, n = 5–8).

(F) Fetal liver acylcarnitine abundances at e17.5

(mean ± SEM, n = 5). For each pair of colored bars,

the left bar represents control fetuses and the right

hatched bar represents Cpt2L�/� fetuses from the

maternal genotype-nutritional combinations rep-

resented in (E).

Statistically significant differences (p < 0.05) for

pairwise comparisons after ANOVA indicated by

letters.

that placental triglyceride content was increased upon fasting

independent of fetal genotype (Figure 1H), concurrent with

increased maternal circulating NEFA during a fast (Figure 1D).

In addition, fasting partially rescued lower liver triglyceride levels

previously observed in Mpc1KI/KI fetuses (Figure 1H) (Bowman

et al., 2016). These data demonstrate that maternal fasting can

affect lipid content within the fetal compartment.


Maternal Lipid Metabolism Drivesthe Fetal Response to FastingTo determine the contribution of maternal

lipid metabolism to the fetal response

to fasting, we used a genetic model of

impaired fasting—liver-specific loss of

mitochondrial b-oxidation of long-chain

fatty acids via Cpt2 (Lee et al., 2016b,

2017). Cpt2 catalyzes an obligate step in

the mitochondrial import of long-chain

fatty acids and is encoded by a single

gene, which makes it a genetically trac-

table target to block mitochondrial

b-oxidation. Mice with liver-specific loss

of Cpt2 (Cpt2L�/�) have massive hepatic

lipid accumulation and impaired ketogen-

esis upon fasting with, surprisingly, no

defects in systemic gluconeogenic ca-

pacity (Lee et al., 2016b). Female mice

homozygous for the floxed Cpt2 allele

(ff) were crossed to males with albumin-

driven Cre recombinase expression

(Cpt2L�/�), and Cpt2L�/� females were

crossed to ff males, such that both

WT (ff) and Cpt2L�/� fetuses could be studied from WT or

mutant Cpt2L�/� dams (Figure 3B). WT and Cpt2L�/� dams

were subjected to 24-h food deprivation from 16.5 to 17.5 dpc,

and, remarkably, Cpt2L�/� dams were able to maintain blood

glucose concentrations to the same level as fasted WT dams

(Figure 2A). Consistent with a defect in liver fatty acid oxidation,

however, Cpt2L�/� dams could not produce bHB during a fast,

and circulating bHB levels in fasted Cpt2L�/� dams were even

lower than fed WT controls (Figure 2A). Fasting also induced

serum NEFA and triglyceride accumulation in Cpt2L�/� maternal

serum (Figure 2A). Altogether, the impaired ketogenesis and

elevated circulating lipid metabolite concentrations of Cpt2L�/�

dams provide a useful model of impaired maternal fasting to

better understand the fetal response to maternal nutrient

deprivation.

Fasting reduced late-gestation Cpt2L�/� fetal body weights to

the same extent as had been observed in WT litters, with no ef-

fect of fetal or maternal genotype (Figure 2B). Importantly, loss of

mitochondrial b-oxidation of long-chain fatty acids in fetal liver

did not alter bHB concentrations as determined by 1H-NMR,

but instead, fetal liver bHB levels reflected maternal circulating

concentrations such that fetuses from fasted Cpt2L�/� dams

had levels as low as fetuses from fed dams of either genotype

(Figure 2C). Fetal liver and placental triglyceride content

increased upon fasting and was similarly regulated independent

of fetal genotype (Figure 2D). These data show that maternal

fasting increases lipid metabolite delivery to the fetus but fetal

liver has little capacity for ketogenesis.

To further determine the effects of altered maternal lipid

metabolism on the fetal response, we characterized the abun-

dance of a class of molecules that can serve as substrates

for the enzymatic function of Cpt2: the long-chain acylcarni-

tines. Cpt2 catalyzes the transacylation of long-chain acylcarni-

tines to become long-chain acyl-CoAs in the mitochondrial

matrix; therefore, loss of Cpt2 would be expected to increase

long-chain acylcarnitine concentrations. We measured both

maternal blood acylcarnitines and fetal liver acylcarnitines

from WT and Cpt2L�/� dams in both the fed and 24-h fasted

states (Figures 2E and 2F). Similar to Cpt2L�/� mice on a keto-

genic diet, pregnant Cpt2L�/� mice exhibited elevated circu-

lating long-chain acylcarnitine concentrations during fasting

and suppressed free carnitine and short-chain acylcarnitines

such as C2 (acetylcarnitine) (Lee et al., 2016b). Additional

short-chain acylcarnitines (C4 and C5), which are derived from

amino acid catabolism, were also downregulated in Cpt2L�/�

circulation (Figure 2E). Long-chain acylcarnitine abundances

were upregulated in fetal liver from fasted WT dams, consistent

with increases in maternal circulating concentrations in

response to fasting, and there were no effects of fetal genotype

(Figure 2F). Interestingly, however, long-chain acylcarnitine con-

centrations in fetal livers from Cpt2L�/� dams were not

increased to the extent that is seen in maternal blood. Increased

maternal oxidation of long-chain acylcarnitines or impaired fetal

fatty acid utilization in litters from Cpt2L�/� dams (independent

of fetal genotype) could contribute to this pattern of acylcarni-

tine levels. These data show that fetal liver fatty acid oxidation

plays a negligible role in maintaining fetal metabolic homeosta-

sis during a fast, but maternal lipid metabolism determines fetal

lipid metabolite concentrations, particularly during an acute

nutrient stress.

Maternal Fasting Response Drives the Fetal LiverTranscriptional ProgramThe loss of hepatic Cpt2 in adult mice results in dyslipidemia

coupled with dramatic increases in the expression of pro-cata-

bolic fatty-acid-responsive genes in the liver and distant organs

such as adipose and kidney (Lee et al., 2016b, 2017). To char-

acterize the putative endocrine impact of impaired maternal

lipid catabolism on the fetal liver transcriptional response, we

performed an RNA sequencing (RNA-seq) analysis of WT fetal

liver derived from fetuses exposed in utero to the metabolic

milieu of either WT or Cpt2L�/� 24-h fasted dams (Figure S1).

In this way, the effects of in utero exposure to impaired maternal

lipid metabolism on the fetal liver transcriptome were deter-

mined independent of fetal genotype. We found metabolic

genes such as enoyl-coenzyme A, hydratase/3-hydroxyacyl co-

enzyme A dehydrogenase (Ehhadh), and pyruvate dehydroge-

nase kinase 4 (Pdk4) were induced, and angiopoietin-like 8

(Angptl8) and acyl-coenzyme A amino acid N-acyltransferase

1 (Acnat1) were suppressed in fetal liver from Cpt2L�/� dams

by 2.5-fold or greater, demonstrating a robust effect of maternal

metabolism on fetal gene expression (Figure 3A). In order to

validate and expand the genes identified, we compared a sub-

set of these genes under all combinations of genetic and envi-

ronmental exposure: WT and Cpt2L�/� genotypes from both

maternal and fetal compartments under both fed and fasted

conditions (Figure 3B). The mRNA abundances of several fast-

ing-regulated genes (acyl-CoA thioesterase 1 (Acot1), Acot2,

and Pdk4) were increased in fetal liver in response to maternal

nutrient deprivation (Figure 3C). While fetuses from fasted WT

dams responded similarly to Cpt2L�/� fetuses from WT fasted

dams, both WT and Cpt2L�/� fetuses from fasted Cpt2L�/�

dams exhibited significantly higher induction of these fasting-

regulated genes than litters from WT dams (Figure 3C). Here,

we have shown that the fetal liver transcriptional response to

maternal fasting was highly dependent on maternal but not fetal

genotype, i.e., the fetal environment was the main determinant

for the transcriptional response independent of the role of fetal

fatty acid catabolism. This suggests that a metabolic or endo-

crine signal altered in fasted Cpt2L�/� dams promotes an exac-

erbated fasting transcriptional program in fetuses exposed to

this intrauterine environment.

The Fetal Response to Fasting Is Ppara-Dependent andRegulated by Maternal MetabolismTo gain a better understanding of the maternal metabolic envi-

ronment and potential metabolite mediators of the endocrine

response that led to changes in fetal hepatic transcription, we

performed global untargeted metabolomics on serum from 24-

h-fasted control and Cpt2L�/� female non-pregnant and preg-

nant mice (Figures 4 and S2). Additionally, we analyzed serum

from 24-h-fasted pregnant Ppara KO mice, Mpc1KI/+ mice, and

Mpc1KI/+; Cpt2L�/� (double knockout [DKO]) dams to identify

conserved metabolic alterations that could account for the fetal

response to maternal fasting in these models (Figure 4A). A prin-

ciple component analysis showed that the serum metabolomes

of pregnant and non-pregnant mice are readily distinguishable

based on pregnancy alone (Figure 4B). In addition, Cpt2L�/�

and Ppara KO dams, despite both having defects in fatty acid

oxidation, exhibit unique serum metabolomes (Figure 4B).

Consistent with the requirement of hepatic fatty acid b-oxidation

for ketone body production, all Cpt2L�/� mice exhibited a dra-

matic reduction in b-hydroxybutyrate, and Ppara KO dams


Figure 3. TheMaternal Response to Fasting

Drives the Fetal Transcriptional Program

(A) Volcano plot of RNA-seq data comparing WT

fetal liver derived from fetuses gestating in either

WT or Cpt2L�/� 24-h-fasted dams. Red points are

significant with a threshold of z R j3j.(B) Experimental scheme.

(C) mRNA abundance of Cpt2 and select fasting-

regulated genes Acot1, Acot2, and Pdk4 in e17.5

liver from all genotypes (mean ± SEM, n = 6).


pairwise comparisons after ANOVA indicated by

letters, ***p < 0.001.

See also Figure S1.

exhibited a significant reduction as well (Figure 4C). Pregnant

mice had higher corticosterone levels than non-pregnant mice,

as expected, but there was no effect of maternal genotype

(Figure 4D). Other pregnancy-specific changes were seen,

such as increased circulating lactate and depleted glycine (Fig-

ure 4D). As we have demonstrated with targeted metabolomics

in Cpt2L�/� mice (Figure 2E), many short-chain acylcarnitine

species were suppressed in Cpt2L�/� but not Ppara KO dams

(Figure 4E). One of the most striking effects seen in both

Cpt2L�/� and Ppara KO dams was the overall increase in circu-

lating lipidmetabolites (Figure S2). For example, most long-chain

free fatty acids, including docosahexaenoic acid (DHA) and

palmitate, were increased in both models (Figures 4E and 4F).

Broad classes of fatty acids and their metabolites were

increased in Cpt2L�/� and Ppara KO dams. This includes known

signaling metabolites such as endocannabinoids and putative

Ppara ligands (Figures 4E and 4F). These data suggest that

increasing signaling lipids in response to maternal nutrient stress

or genetic perturbations can signal fromdam to fetus to alter fetal

liver transcriptional programming.

The metabolomics analysis demonstrated an induction of

circulating Ppara ligands, and many of the genes observed to

be transcriptionally regulated in fetal liver in response to impaired

mitochondrial pyruvate metabolism (Bowman et al., 2016) or

in response to maternal fasting are canonical Ppara target

genes. Therefore, we next turned our attention to mice with a

deletion of the lipid-dependent transcriptional regulator and

so-called master regulator of fatty acid catabolism, Ppara.

Upon subjecting dams to 24-h food deprivation, e17.5 fetuses

with heterozygous or homozygous Ppara deletion fromPpara�/�


dams exhibited a decrease in body

weight comparable to fasted WT litters

(Figure 5A). Moreover, Ppara+/� and

Ppara�/� fetuses from Ppara�/� dams

were not different in size than the same

fetal genotypes from Ppara+/� dams.

Therefore, the loss of Ppara in the

maternal or fetal compartment did not

affect fetal growth, similar to what was

observed for Cpt2L�/� mice (Figure 2B).

To examine the dependence of the

fetal transcriptional response to maternal

Ppara, we performed RNA-seq on

Ppara+/� fetal livers that were derived from fasted Ppara+/�

or Ppara�/� dams. Similar to Cpt2L�/� dams, the loss of

maternal Ppara and induction of lipid signaling molecules in

maternal circulation was sufficient to induce Ppara-dependent

genes in Ppara+/� fetal livers (Figure 5B). Additionally, we per-

formed RNA-seq analysis on Ppara�/� fetal livers from

Ppara�/� dams to understand the dependence of these

changes on fetal Ppara. We identified many genes that were

induced upon fasting in Ppara+/� fetuses gestating in Ppara�/�

dams that were dependent upon fetal Ppara signaling—that is,

genes with lower expression in Ppara�/� fetuses (Figures 5C

and S1). To expand and validate these changes, we examined

fasting-regulated gene induction in fed and fasted WT,

Ppara+/�, and Ppara�/� fetal livers from WT, Ppara+/�, and

Ppara�/� dams (Figure 5D). The genes shown in Figure 5D

demonstrated Ppara-dependent fasting induction in that

Ppara�/� livers failed to induce expression of these genes

(Acot1, Acot2, Pdk4, Ehhadh, Apoa4, and Epn3) upon fasting.

While the fetal liver transcriptional response to fasting requires

fetal Ppara, the effect of the intrauterine environment is also

critical for eliciting these responses, as the fetal response to

fasting in Ppara�/� dams was most robust (Figure 5D). These

data again suggest that maternally derived lipid signals from

models of impaired fatty acid catabolism can potentiate the

fetal liver response to maternal nutrient deprivation and that

the fetal response is at least partially Ppara dependent. This

is consistent with the Cpt2L�/� model in which fetal liver from

fasted Cpt2L�/� dams had higher liver expression of fasting-

regulated transcripts than fetal liver from fasted WT control

dams (Figure 3).

Figure 4. Untargeted Metabolomics Reveals Pregnancy- and Genotype-Regulated Serum Metabolites

(A) Unsupervised clustering of all serum metabolites.

(B) Principle component analysis of the serum metabolome data.

(C) Relative concentrations of serum b-hydroxybutyrate in pregnant (17.5 dpc) and non-pregnant mice of the genotypes indicated (mean ± SEM, n = 6).

(D) Pregnancy-specific changes in metabolites.

(E) Lipid metabolite changes as a consequence of genotype.

(F) Broad classes of lipids derived from the fatty acid palmitate that are regulated by genotype (mean ± SEM, n = 6).

Statistically significant differences (p < 0.05) for pairwise comparisons after ANOVA compiled in Table S2.

See also Figure S2.


Figure 5. Ppara-Dependent Fetal Response

to Maternal Fasting Is Potentiated by

Impaired Maternal Lipid Metabolism

(A) e17.5 body weights from WT dams, Ppara�/�

dams carrying heterozygous or homozygous

Ppara deletion litters (shaded in yellow), or all three

fetal genotypes (WT, +/�, and �/�) from Ppara+/�

intercrosses (shaded in orange). Circles are from

fed litters and squares are from fasted litters.

(B) Volcano plot of RNA-seq data comparing

Ppara+/� fetal liver derived from fetuses gestating

in either Ppara+/� or Ppara�/� 24-h-fasted dams.

(C) Volcano plot of RNA-seq data comparing

Ppara�/� fetal liver from Ppara�/� 24-h-fasted

dams to Ppara+/� fetuses from Ppara+/� dams.

Red points are significant with a threshold of

z R j3j.(D) mRNA abundance of fasting-regulated genes

Acot1, Acot2, Pdk4, Ehhadh, Apoa4, and Epn3 in

fetal liver, same genotypes as described in (A).

Open bars are for fed samples; filled bars are for

fasted samples (mean ± SEM, n = 6).


pairwise comparisons after ANOVA compiled in

Table S2.

Combined Deficiencies of Carbohydrate and Fatty AcidMetabolism further Potentiate the Fetal Response toMaternal FastingTo understand the effects ofmaternal lipidmetabolism on fetuses

with impaired glucose metabolism, we crossed Mpc1KI/+ and

Cpt2L�/� mice to generate dams with defective hepatic fatty

acid oxidation and heterozygosity for Mpc1 deficiency (Cpt2L�/�;Mpc1KI/+). These dams carried fetuses that were either WT or

Mpc1 deficient (Mpc1KI/KI) frommating toCpt2f/f;Mpc1KI/+males.

Mpc1 heterozygosity did not further potentiate defects in

Cpt2L�/� maternal serum chemistry (Figures 6A and 6B).


Following a 24-h fast Cpt2L�/�; Mpc1KI/+

17.5 dpc dams maintained normal fasting

blood glucose, exhibited elevated NEFA

and triglycerides, and showed an absence

of bHB, as expected (Figure 6A). Addition-

ally, 24-h-fasted Cpt2L�/�; Mpc1KI/+ 17.5

dpc dams exhibited decreased blood

carnitine and short-chain acylcarnitines

with elevated long-chain acylcarnitines

similar to Cpt2L�/� dams (Figure 6B).

These data show that late-gestation

Cpt2L�/�; Mpc1KI/+ dams exhibit a similar

metabolic dysfunction to Cpt2L�/� dams,

including a loss in bHB and increased

circulating lipids, consistent with global

untargeted metabolomics (Figure 4).

Next, wemeasured the lactate concen-

tration in WT and Mpc1KI/KI e17.5 fetal

brains from Mpc1KI/+ dams with (Cpt2f/f;

Mpc1KI/+) and without (Cpt2L�/�;Mpc1KI/+) hepatic fatty acid oxidation

following a 24-h fast. As expected, the

loss of maternal hepatic fatty acid oxidation eliminated bHB

accumulation in both WT and Mpc1KI/KI fetal liver (Figure 6D).

The loss of ketone body production in Cpt2L�/�; Mpc1KI/+

dams modestly affected lactate accumulation in Mpc1KI/KI

e17.5 fetal brains (Figure 6C). By profiling fetal liver metabolites

by 1H-NMR, the loss of maternal and fetal Mpc1, in addition to

the loss of maternal bHB, resulted in significant suppressions

in glucose and tricarboxylic acid (TCA) cycle intermediates (Fig-

ure 6D). The duel impairment of maternal and fetal carbohydrate

and lipid mitochondrial metabolism created significant meta-

bolic limitations for the developing fetus.

Figure 6. Combined Deficiencies of Carbohydrate and Fatty Acid Oxidation Further Potentiate the Fetal Response to Maternal Fasting

(A) Maternal blood glucose, blood lactate, and serum b-hydroxybutyrate, NEFAs, and triglyceride (TG) concentrations after 24-h fast from 16.5 to 17.5 dpc (mean

± SEM, n = 5–8).

(B) Maternal 24-h-fasted blood acylcarnitine abundances shown relative to control dam (mean ± SEM, n = 5).

(C) Fetal brain lactate concentrations measured enzymatically (mean ± SEM, n = 5).

(D) Steady-state metabolite abundances from e17.5 fetal liver, determined by 1H-NMR. Normalized to protein content and shown relative to WT fetus from

MpcKI/+ dam (mean ± SEM, n = 5).

(E) Fetal liver transcriptional response determined by qRT-PCR (mean ± SEM, n = 5-6).

Statistically significant differences (p < 0.05) for pairwise comparisons after ANOVA indicated by letters.

Finally, we assayed fetal transcriptional programming in the

liver of fetuses exposed to these disparate maternal environ-

ments. As we have shown before, Cpt2L�/� dams potentiate

the fetal response to fasting. Consistent with the increased

metabolic dysfunction in the fetal liver, the loss of one copy of

Mpc1 in Cpt2L�/� dams greatly exacerbated the fetal response

to fasting as Acot1, Acot2, Cpt1b, and Pdk4 were further

increased in fetal livers of Cpt2L�/�; Mpc1KI/+ dams (Figure 6E).

In fact, fetal liver Pck1 expression from fasted Cpt2L�/�;Mpc1KI/+ dams was induced �40-fold over fed WT controls. As

we showed previously, fetal loss of Mpc1 further potentiated

this transcriptional response independently by �2-fold. These

data show that the compound loss of Mpc1 and Cpt2 greatly

enhanced the fetal transcriptional response to maternal fasting


above what was observed when either was deleted alone. Alto-

gether, the metabolic and transcriptional changes observed in

these genetic models provide evidence for robust metabolic

communication between mother and fetus, particularly in the

context of nutrient restriction.

DISCUSSION

The fetus is thought to utilize nutrients much like the adult

mammalian brain; that is, it preferentially utilizes glucose but

can switch to ketone bodies following prolonged fasting (Girard

et al., 1977). Mutations in the MPC result in pyruvate and lactate

acidosis in mouse models and human inborn errors that are not

compatible with sustained independent life (Bricker et al., 2012;

Herzig et al., 2012). In utero, these effects can be somewhat miti-

gated by maternal metabolite clearance, but MPC mutations

result in developmental defects (Bowman et al., 2016; Vander-

perre et al., 2016). Incredibly, simply feeding pregnant mice a

ketogenic diet from 8.5 dpc can rescue many of the develop-

mental defects seen in MPC mutant fetuses (Sussman et al.,

2013; Vanderperre et al., 2016). As the ketogenic diet is a fasting

mimetic, we sought to understand the effects of maternal fasting

on fetal metabolism and have shown that fasting is sufficient to

reverse many of the metabolic alterations in MPC mutant fe-

tuses. This is remarkable given the putative requirements of

the fetus for glucose and suggests an incredible and previously

unrecognized metabolic flexibility in the rapidly growing fetus.

Due to the incredible demand for glucose during gestation,

fasting during pregnancy results in an accelerated starvation

response that is characterized by an earlier and more exagger-

ated shift to ketone body utilization (Boden, 1996; Freinkel,

1980). We have demonstrated that ketone bodies derived from

maternal liver fatty acid oxidation, in combination with increased

serum concentrations of triglyceride, phospholipids, and NEFAs,

may help mediate this metabolic adaptation. Levels of theseme-

tabolites increase over the course of gestation, and circulating

triglyceride levels, for example, increase more than 250% by

the end of pregnancy, which is almost twice the post-prandial

concentration in a non-pregnant individual (Darmady and Postle,

1982; Haggarty, 2010). The increase in concentrations of these

lipids and lipid-derived metabolites is so dramatic that overnight

fasting (14–18 h) in normal late-term pregnancy leads to serum

metabolite concentrations that rival the effects of 2–3 days of

starvation in non-pregnant individuals (Boden, 1996; Metzger

et al., 1982). Fasting during pregnancy results in an earlier than

normal shift from glucose to fat and ketone utilization by

maternal tissues to spare glucose and amino acids for fetal up-

take (Metzger et al., 1982), and we have shown that mitochon-

drial metabolism is an important regulator of these changes in

substrate utilization.

Deleting Cpt2 and therefore fatty acid b-oxidation from the

liver renders mice incapable of generating sufficient ketone

bodies and increases serum lipid levels (Lee et al., 2016b,

2017).We took advantage of thismodel to understand the contri-

bution of maternal lipid metabolism to the fetal fasting response,

including the requirement for ketone body delivery to the fetus.

We have previously shown that adult Cpt2L�/� mice exhibit an

incredible induction of pro-catabolic genes in their livers


following a fast (Lee et al., 2016b). Here, we have shown that fetal

liver b-hydroxybutyrate levels closely reflect maternal circulating

concentrations, and the loss of fatty acid b-oxidation does

not affect transcriptional programming in a fetal-autonomous

manner. However, the loss of Cpt2 frommaternal liver is critically

important for fetal ketone body delivery and fetal transcriptional

programming in response to fasting.

Following a fast, adult Cpt2L�/� mice induce a pro-catabolic

endocrine program in non-hepatic tissues to maintain systemic

energy homeostasis (Lee et al., 2016b). Similarly, defects in

maternal hepatic fatty acid oxidation or a loss of maternal Ppara

generates an endocrine signal that affects fetal liver transcrip-

tional programming. Classic maternal-fetal endocrine loops

have been identified, such as glucocorticoid signaling, that are

critical for fetal development, maturation, and parturition during

late gestation (Liggins, 1968; Liggins et al., 1967; Rando et al.,

2016). We have shown that maternal nutrient deprivation or de-

fects in maternal lipid catabolism can signal to the fetus presum-

ably through the dramatic increase in circulating maternal fatty

acid Ppara ligands. Untargeted and targeted metabolomics re-

vealed broad classes of signaling lipids that are induced,

including Ppara ligands and endocannabinoids such as acyle-

thanolamines and acyltaurines. In contrast to proteinaceous hor-

mones, many of the hydrophobic metabolites that we identified

would be predicted to cross the hemochorial placenta, although

detailed pharmacokinetics have not been performed. While we

have shown that the loss of fetal Ppara abrogates much of the

transcriptional response, there is clearly Ppara-independent

regulation as well. This may bemediated by other classes of me-

tabolites, for example, the endocannabinoids (Correa et al.,

2016). The loss of these critical metabolic nodes in central car-

bon metabolism affects many pathways, as we have shown in

the metabolomics analysis, and some of the transcriptional ef-

fects may be quite indirect by affecting placental metabolism

or transport.

The acute fasting paradigm employed here should be con-

trasted with chronic nutritional deprivation caused by poor nutri-

tion, limited access to food, or placental insufficiency. Acute

fasting is not an absence of nutrients but a dramatic shift in the

type of nutrients available, many being lipids or lipid-derived me-

tabolites. The types of genes affected by fasting and potentiated

by a lack of maternal lipid catabolism suggest that the fetus is

accelerating its ability to utilize oxidative metabolism. The fetus

increases the expression of genes involved in oxidative meta-

bolism throughout pregnancy as oxygen becomes more avail-

able and to prepare the fetus for the consumption of postpartum

milk fat. By our model, we propose that fasting in late gestation

may hasten this progression in preparation for parturition.

Together, our genetic mouse models of impaired mitochon-

drial pyruvate and fatty acid metabolism demonstrate the impor-

tance of mitochondrial oxidative metabolism in regulating both

the maternal and fetal responses to nutrient deprivation. Despite

the nutritionally sensitive nature of this stage in mammalian

development, there exists remarkable metabolic flexibility and

crosstalk among these major pathways of central carbon meta-

bolism. Maternal-fetal metabolic communication helps ensure

adequate nutrient exchange and is particularly important during

times of nutrient limitation. Miscommunication may result in

metabolic complications during pregnancy including gestational

diabetes, and promoting healthy maternal-fetal metabolic

communication may prove an important therapeutic approach

for metabolic disorders of pregnancy.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Generation of Genetic Mouse Models

B Ethical Statement

B Cell Culture

d METHOD DETAILS

B Serum and Tissue Metabolite Measurement

B Immunoblotting

B Quantitative Real-Time PCR

B RNA Sequencing and Analysis

B Acylcarnitine Analysis of Blood and Liver

B Pathway Analysis

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND CODE AVAILABILITY

B Accession Numbers

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

celrep.2019.09.053.

ACKNOWLEDGMENTS

This work was supported in part by NIH grants R01NS072241 and

R01DK116746 to M.J.W. and R01NS110808 and R01NS111230 to S.S.

C.E.B. was supported in part by a fellowship from the American Heart Associ-

ation (15PRE25090309).

AUTHOR CONTRIBUTIONS

Conceptualization, C.E.B. and M.J.W.; Methodology, C.E.B., E.S.S.A., and

M.J.W.; Investigation, C.E.B., E.S.S.A., D.M.S., S.S., andM.J.W.; Formal Anal-

ysis, all authors; Visualization, C.E.B., E.S.S.A., K.C., and M.J.W.; Resources,

S.S. and M.J.W.; Writing – Original Draft, C.E.B. and M.J.W; Writing – Review-

ing & Editing, all authors; Funding Acquisition, S.S. and M.J.W.

DECLARATION OF INTERESTS

The authors have no competing financial interests.

Received: May 2, 2019

Revised: August 5, 2019

Accepted: September 18, 2019

Published: October 29, 2019

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Mpc1, rabbit polyclonal Bowman et al., 2016 N/A

Mpc2, rabbit polyclonal Bowman et al., 2016 N/A

Hsc70, heat shock chaperone 70, mouse monoclonal Santa Cruz Cat#sc-7298; RRID: AB_627761

Deposited Data

RNaseq data This paper GEO GSE129368

Experimental Models: Cell Lines

Mpc1 deletion mouse embryonic fibroblasts (MEFs) Bowman et al., 2016 N/A

Experimental Models: Organisms/Strains

Mouse: Mpc1 knock-in (KI) hypomorphic allele in C57BL/6J mice Bowman et al., 2016 N/A

Mouse: Cpt2 floxed allele in C57BL/6J mice with Albumin-Cre transgene Lee et al., 2016b, 2017 N/A

Mouse: Ppara knock-out (KO) in C57BL/6J mice Lee et al., 1995; The Jackson

Laboratory

JAX: 008154

Oligonucleotides

See Table S3 N/A N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

All unique/stable reagents generated in this study are available from the Lead Contact without restriction. Further information and

requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Michael Wolfgang

([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Generation of Genetic Mouse ModelsMpc1 knock-in mice were generated by targeting loxP sequences to introns flanking exons 3 to 5 of the mouse Mpc1 gene and

targeting a transcriptional reporter construct to the 30 untranslated region of Mpc1 by homologous recombination in C57BL/6J

embryonic stem cells by standard methods, as previously described (Bowman et al., 2016). A conditional knock-out (KO) allele of

mouse Cpt2, carnitine palmitoyltransferase 2, was generated by the same approach (Gonzalez-Hurtado et al., 2017, 2018; Kim

et al., 2017; Lee et al., 2015, 2016a, 2016b; Nomura et al., 2016). To generate mice with a liver-specific loss of mitochondrial fatty

acid b-oxidation, Cpt2 floxed mice were crossed to Albumin-Cre (Alb-Cre) transgenic mice (Postic et al., 1999) to generate animals

(Cpt2L�/�) that cannot oxidize long-chain fatty acids in hepatocytes (Lee et al., 2016b, 2017). PPARa�/�mice (stock number 008154)

were obtained on a C57BL/6J background from The Jackson Laboratory and were crossed to wild-type (WT) mice for studies of het-

erozygotes (Lee et al., 1995).

All mutant mice and littermate controls were housed in a facility with ventilated racks on a 14h light/10h dark cycle with ad libitum

access to a standard rodent chow (2018SX Teklad Global, 18% protein). For timed matings, the presence of a copulatory plug in

the morning was designated as 0.5 days post coitum (dpc), and pregnant females were housed separately. Female mice pregnant

with their first litter were 12-18 weeks old at the end of late gestation studies. Non-pregnant controls were age matched. For 24h

fasting experiments, food was removed from pregnant dams at 16.5 dpc between 13:00-15:00, and tissues were collected 24h later

at 17.5 dpc.

Ethical StatementAll procedures were performed in accordance with the NIH’s Guide for the Care and Use of Laboratory Animals and under the

approval of the Johns Hopkins Medical School Animal Care and Use Committee.

Cell CultureMouse embryonic fibroblasts (MEFs) were obtained from timed pregnant dams by standard methods. Primary MEFs were main-

tained in DMEM (25mM glucose) supplemented with 10% fetal bovine serum and 1% pen/strep antibiotic (Invitrogen) at 37�C in a

Cell Reports 29, 1299–1310.e1–e3, October 29, 2019 e1


humidity-controlled incubator at 10%CO2. For1H-NMR of cell extracts, cells were switched to serum-free DMEM containing 2.5mM

glucose, 2.5mMpyruvate, and 2mMglutamine in the presence or absence of 1mMD,L-b-hydroxybutyrate (Sigma-Aldrich, H6501) for

24h before collection of cell extracts for steady-state metabolite measurements as described above.

METHOD DETAILS

Serum and Tissue Metabolite MeasurementSerum metabolites were determined by enzymatic, colorimetric assays, according to the manufacturer’s instructions: b-hydroxybu-

tyrate (Stanbio B-HB LiquiColor Assay, EKF Diagnostics, Boerne, TX), NEFA (NEFA-HR(2), Wako Diagnostics, Richmond, VA), tri-

glyceride (TR0100, Sigma-Aldrich, St. Louis, MO). Placental and liver triglyceride content was also determined by enzymatic

measurement after chloroform: methanol (2:1 (v/v)) extraction, drying, and resuspension in 3:1:1 (v/v/v) t-butanol: methanol: Triton

X-100. Triglyceride levels were normalized to protein content as determined by bicinchoninic acid (BCA) assay (Thermo Fisher Sci-

entific). Blood glucose and lactate were measured with a Nova Max Plus glucometer and Lactate Plus meter (Nova Biomedical, Wal-

tham, MA). Tissue lactate concentrations were determined enzymatically after homogenizing frozen tissues in lactate assay buffer on

ice and were normalized to protein content (MAK065, Sigma-Aldrich, St. Louis, MO).

For 1H-NMR of tissue and serum metabolites, extraction was performed as previously reported (Lee et al., 2017). For 1H-NMR of

cell extracts, mouse embryonic fibroblasts (MEFs) were washed with ice-cold PBS and scraped into ice-cold methanol and snap

frozen in liquid nitrogen before two additional freeze-thaw rounds with methanol extraction (Bowman et al., 2017). Cell extracts

were subjected to a final spin at 15,000 g for 1min at 4�C, then the supernatant was dried under vacuum and the extract resuspended

in 20 mM phosphate buffer, pH 7.4 ± 0.1, with 0.1 mM TMSP as an internal reference and 0.1 mM sodium azide. 1H spectra were

recorded on a Bruker Avance III 500MHz (Bruker Instruments, Germany) NMR spectrometer, operating at 499.9MHz and equipped

with room temperature quadruple nuclei probe. Typical 1H spectra were acquired using presaturation solvent suppression pulse

sequence (noesyprld). Acquisition parameters were set as follows; spectral width of 8012.820 with 64K data points, 512 scans,

with a relaxation delay of 7 s for a total collection time of 1.14h. Samples were automatically tuned and matched, and shimmed

to TMSP signal. Spectra were exported into Bruker format and were processed with Chenomx NMR Suit 8.2 Professional (Chenomx

Inc, Edmonton, Alberta, Canada). TMSP signal (0.0 ppm) was used as a reference peak, spectra were manually phase corrected and

spline function was applied for the baseline correction. Metabolites were profiled and quantified using built-in Chenomx 500MHz

library. Metabolite concentrations were normalized to protein content. Untargeted metabolomics was done as previously described

(Bowman et al., 2016).

ImmunoblottingTissue lysates for SDS-PAGEwere prepared by homogenization of tissue in RIPA buffer (50mMTris-HCl, pH 7.4, 150mMNaCl, 1mM

EDTA, 1%Triton X-100, 0.25%deoxycholate) with protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche),

followed by pelleting of insoluble debris at 13,000 g for 15 min at 4�C. Protein concentrations of lysates were determined by BCA

assay (Thermo Scientific), and 30mg of lysate was separated by Tris-Glycine SDS-PAGE (15% polyacrylamide), unless otherwise

noted. Proteins were transferred to PVDF membranes (Immobilon), blocked in 5% nonfat-milk TBST, and incubated with primary

antibodies overnight. Primary antibodies used include: We generated and validated affinity-purified rabbit polyclonal antibodies

against mouse Mpc1 and Mpc2 (Bowman et al., 2016); heat shock chaperone 70 (Hsc70) mouse monoclonal at 1:1000 (Santa

Cruz sc-7298); Cy3-conjugated anti-mouse (Invitrogen) or Cy5-conjugated anti-rabbit (Invitrogen) secondary antibodies at 1:1500,

or horseradish peroxidase (HRP)-conjugated anti-rabbit (GE Healthcare NA934V) secondary antibodies at 1:2000 were incubated

with washed membranes, and proteins were visualized with Amersham Prime enhanced chemiluminescent substrate (GE Health-

care) or epifluorescence on an Alpha Innotech MultiImage III instrument. Protein abundance was quantified using Alpha Innotech

FluorChem Q software (Santa Clara, CA) and was normalized to Hsc70 expression.

Quantitative Real-Time PCRTotal RNA was extracted using Trizol reagent, per manufacturer’s recommendations (Life Technologies) and was further purified

using the RNeasy Mini kit (QIAGEN). RNA was quantified by NanoDrop, and cDNA was synthesized using random primers and

MultiScribe High-Capacity cDNA reverse transcription kit (Applied Biosystems). RT-PCR was performed using 10 ng of template

cDNA in a 20mL reaction using Bio-Rad SsoAdvanced SYBR Green master mix with primers specific to the genes of interest (Table

S3). All PCR reactionswere carried out in a Bio-RadCFXConnect Thermocycler andwere concludedwith amelt-curve determination

step. Expression data from mouse tissues was normalized to the average Ct values for 4 reference genes: 18S, Rpl22, b-actin, and

Gapdh, and data are expressed as 2-dCt and shown relative to wild-type or fed controls, as appropriate.

RNA Sequencing and AnalysisTotal RNA was extracted from frozen liver tissue from wild-type, Ppara+/�, or Ppara�/� e17.5 mice (n = 4, 2 male, 2 female)

from fasted WT, Cpt2L�/�, Ppara+/�, or Ppara�/� dams using Trizol reagent and purified using the RNeasy Mini kit (QIAGEN) as

described above. RNA quality was assessed, and Illumina TruSeq stranded mRNA library was prepared for sequencing single

75bp reads using an Illumina NextSeq 500 sequencing platform. CLC Genomics Server v10.0.1 was used to assemble and align

e2 Cell Reports 29, 1299–1310.e1–e3, October 29, 2019

reads to the mouse reference genome GCRm38.p6, and determine FPKM. Log2 transformed FPKM values were quantile normalized

with Partek Genomics Suite v7.0. Differential expression between samples was calculated using one-way ANOVA. Significance

threshold was set as a binned z-score of 3 for transcripts that were found in minimum 2 animals per comparison genotype, where

z = ðXgene � Xpopulation =spooledÞ. Use of z-score threshold accounts for total variance (s2) in RNA levels between genotypes. Volcano

plots of NCBI protein coding genes were generated using GraphPad Prism.

Acylcarnitine Analysis of Blood and LiverAcylcarnitine abundances were determined from dried blood spot samples (Chace et al., 1997; Sandlers et al., 2012) or from frozen

liver, as previously described (Lee et al., 2016b, 2017). Tissues acylcarnitine concentrations were normalized to frozen tissue weight.

Briefly, samples were methanol extracted and butylated in the presence of acid, further extracted, and analyzed on an API 3200 (AB

SCIEX) operated in positive ion mode, with a precursor ion scan for m/z 85, which is a characteristic product ion of butyl ester acyl-

carnitine species.

Pathway AnalysisPathway analysis was carried out using quantile normalized RNA-seq values. Indicated ANOVA values were taken from the one-way

ANOVA described in RNA sequencing and analysis. Genotypic transcript levels were calculated by normalizing an individual FPKM

count to the average count for a given gene. The PCA plot was generated using theweb-based data analysis tool MetaboAnalyst. The

normalized expression heatmap was generated from the genes with the 200 lowest grand p values from the one-way ANOVA. The

Ppar⍺ target heatmapwas generated using the log2 fold change over control values as determined by one-way ANOVA. Target genes

were obtained from PPARgene (Fang et al., 2016). Heatmaps were visualized using GraphPad Prism 8. KEGG pathway analysis was

carried out using DAVID on differentially expressed transcripts relative to control genotypes as determined by the significance

threshold of zR j3j in a one-way ANOVA (Huang da et al., 2009). Metabolomics pathway analysis was performed viaMetaboAnalyst.

(Chong et al., 2018).

QUANTIFICATION AND STATISTICAL ANALYSIS

One-way and two-way ANOVA were used to evaluate statistical significance for quantitation of body weights and steady-state

metabolite concentrations. For global serum metabolomics, unsupervised hierarchical clustering and principle component analysis

(PCA) were conducted using the web-based metabolomics data analysis tool MetaboAnalyst (Chong et al., 2019).

DATA AND CODE AVAILABILITY

Accession NumbersThe Gene Expression Omnibus (GEO) accession number for the RNA-seq data reported in this paper is GSE129368.

Cell Reports 29, 1299–1310.e1–e3, October 29, 2019 e3

Cell Reports, Volume 29

Supplemental Information

Maternal Lipid Metabolism Directs Fetal Liver

Programming following Nutrient Stress

Caitlyn E. Bowman, Ebru S. Selen Alpergin, Kyle Cavagnini, Danielle M. Smith, SusannaScafidi, and Michael J. Wolfgang

SUPPLEMENTARY FIGURE LEGENDS Figure S1. Fetal liver transcriptional adaptation to maternal fasting, related to Figures 3 and 5. Maternal and fetal genotype combinations indicated above A. (A) PCA plot of RNA-seq data from fetal livers. (B) Heatmap of top 200 differentially expressed transcripts. Values indicate expression levels normalized to average FPKM count for a given transcript. (C) KEGG pathway analysis for differentially expressed transcripts relative to control genotypes with a significance threshold of z ≥ |3|. Red bars correspond to pathways identified in upregulated gene, while blue bars correspond pathways identified in downregulated transcripts. Enrichment score is indicated by log10(p-value). (D) RNA-seq expression levels for Ppar⍺ target transcripts in fetal liver, as determined by normalizing genotype FPKM values to the average count for a given transcript. Sample identities indicated above graphs, with control genotypes indicated by arrows. (E) Heat map of differentially expressed known Ppar⍺ target genes in fetal liver. Values indicate the log2 fold change in expression over control genotypes. Figure S2. Pathway analysis for maternal serum metabolomics, related to Figure 4. Pathway analysis showing p-values for enrichment across comparisons. Pathway analysis was done via metaboanalyst. Table S1. Excel file of raw data for maternal serum, maternal liver, and fetal liver metabolomics. Table S2. Excel file of statistical analysis of metabolites from Fig. 4-5.

Nor

mal

ized

exp

ress

ion

leve

ls

Pdk4

2

1

0

-1

Plin22

1

0

Fabp1

1

0

-1

Ehhadh

2

3

1

0

-1

-2

Acot1

1

0

-1

-2

Acaa1b2

0

-2

-3

1

1

maternal Pparα-/- ; fetal Pparα+/-

maternal Pparα-/- ; fetal Pparα-/-

maternal Pparα+/- ; fetal Pparα-/+

maternal Cpt2L-/- ; fetal Cpt2WT

maternal Cpt2WT; fetal Cpt2WT

D

EhhadhPdk4Acot1Plin2Acaa1bAngptl4AhrGptCpt1bSlc25a20Creb3l3Acox1Fabp1Hmgcs2Apoa5RetsatGys2AcadmGpd1Rbp2Sema6bIl1rnPdzk1Lxra

20-2log2 fold change over control

Pparα target genes

matern

al Pparα

-/- ; fet

al Pparα

+/-

matern

al Cpt2

L-/- ; fet

al Cpt2

WT

matern

al Pparα

-/- ; fet

al Pparα

-/-

E

-10 -8 -6 -4 -2 0 2 4

Tryptophan metabolismABC transporters

Tyrosine metabolismMetabolic pathways

PPAR signaling pathwayPeroxisome

Biosynthesis of unsaturated fatty acids

KEGG Pathway log10(p-value)

maternal Pparα-/- ; fetal Pparα+/- maternal Pparα+/- ; fetal Pparα+/-

KEGG Pathway log10(p-value)-15 -12 -9 -6 -3 0 3

Fatty acid metabolismPPAR signaling pathway

Fatty acid degradationMetabolic pathways

PeroxisomeRetinol metabolism

p53 signaling pathway

maternal Pparα-/- ; fetal Pparα-/- maternal Pparα+/- ; fetal Pparα+/-

KEGG Pathway log10(p-value)-3 0 3 6 9 12 15

Retinol metabolismTGF-beta signaling pathway

Fatty acid degradationFat digestion and absorption

PPAR signaling pathwayViral carcinogenesis

AlcoholismSystemic lupus erythematosus

maternal Cpt2L-/- ; fetal Cpt2WT maternal Cpt2WT ; fetal Cpt2WT

Upregulated genesDownregulated genesC

PC1 (28.8% of variation)

PC2

(15.

8% o

f var

iatio

n)

-150 -100

-100

100

-200

-50 50 100 1500

0

Normalized expression levels-2 -1 0 1 2

Genotype

A

B

maternal Pparα-/- ; fetal Pparα+/-

maternal Pparα-/- ; fetal Pparα-/-

maternal Pparα+/- ; fetal Pparα-/+

maternal Cpt2L-/- ; fetal Cpt2WT

maternal Cpt2WT ; fetal Cpt2WT

GenotypeFigure S1

4

Color Key

0.00 0.01 0.02 0.03 0.04 0.05

Fatty acid biosynthesis

Primary bile acid biosynthesis

Nicotinate and nicotinamide metabolism

Biosynthesis of unsaturated fatty acids

Phenylalanine metabolism

Glyoxylate and dicarboxylate metabolism

Phenylalanine, tyrosine and tryptophan biosynthesis

Fatty acid elongation in mitochondria

Fatty acid metabolism

Arachidonic acid metabolism

Biotin metabolism

Synthesis and degradation of ketone bodies

Aminoacyl-tRNA biosynthesis

Linoleic acid metabolism

Glycine, serine and threonine metabolism

alpha-Linolenic acid metabolism

Lysine degradation

Cysteine and methionine metabolism

Sphingolipid metabolism

D-Arginine and D-ornithine metabolism

Vitamin B6 metabolism

Glycerolipid metabolism

Tryptophan metabolism

Arginine and proline metabolism

Taurine and hypotaurine metabolism

Fructose and mannose metabolism

Pentose phosphate pathway

0.00 0.01 0.02 0.03 0.04 0.05

Fatty acid metabolismPhenylalanine metabolism

Biosynthesis of unsaturated fatty acidsSynthesis and degradation of ketone bodies

Phenylalanine, tyrosine and tryptophan biosynthesisNicotinate and nicotinamide metabolism

Fatty acid biosynthesisFatty acid elongation in mitochondria

D-Arginine and D-ornithine metabolismArachidonic acid metabolism

Glycerophospholipid metabolismAminoacyl-tRNA biosynthesis

Glycine, serine and threonine metabolismArginine and proline metabolism

Linoleic acid metabolismPyrimidine metabolism

alpha-Linolenic acid metabolismTerpenoid backbone biosynthesis

Sulfur metabolismPantothenate and CoA biosynthesis

Galactose metabolismAmino sugar and nucleotide sugar metabolism

Cysteine and methionine metabolismFructose and mannose metabolism

Taurine and hypotaurine metabolismPurine metabolism

beta-Alanine metabolismPrimary bile acid biosynthesis

Valine, leucine and isoleucine degradationNitrogen metabolism

Valine, leucine and isoleucine biosynthesisButanoate metabolism

Glycosylphosphatidylinositol(GPI)-anchor biosynthesisAlanine, aspartate and glutamate metabolism

Histidine metabolismTyrosine metabolism

Sphingolipid metabolismUbiquinone and other terpenoid-quinone biosynthesis

Steroid biosynthesisBiotin metabolism

Lysine degradationGlyoxylate and dicarboxylate metabolism

Methane metabolismCyanoamino acid metabolism

Thiamine metabolismGlutathione metabolism

Steroid hormone biosynthesisAscorbate and aldarate metabolism

Inositol phosphate metabolismSelenoamino acid metabolism

Tryptophan metabolismGlycerolipid metabolism

Pregnant Wt vs Pregnant Cpt2 Non-Pregnant Wt vs Non- Pregnant Cpt2

0.0 0.2 0.4 0.6 0.8 1.0

Fructose and mannose metabolismHistidine metabolism

Galactose metabolismNicotinate and nicotinamide metabolism

Ascorbate and aldarate metabolismInositol phosphate metabolism

Amino sugar and nucleotide sugar metabolismArginine and proline metabolism

Lysine degradationPentose and glucuronate interconversions

Terpenoid backbone biosynthesisTryptophan metabolism

Pantothenate and CoA biosynthesisPyrimidine metabolismVitamin B6 metabolism

Glycerophospholipid metabolismbeta-Alanine metabolism

Fatty acid biosynthesisSulfur metabolismPurine metabolism

Lysine biosynthesisFatty acid elongation in mitochondria

Glycosylphosphatidylinositol(GPI)-anchor biosynthesisPhenylalanine metabolism

Aminoacyl-tRNA biosynthesisTyrosine metabolism

Ubiquinone and other terpenoid-quinone biosynthesisStarch and sucrose metabolism

Biosynthesis of unsaturated fatty acidsD-Glutamine and D-glutamate metabolism

Cysteine and methionine metabolismLinoleic acid metabolism

Phenylalanine, tyrosine and tryptophan biosynthesisSteroid biosynthesis

Thiamine metabolismFatty acid metabolism

Primary bile acid biosynthesisSteroid hormone biosynthesis


Nitrogen metabolismBiotin metabolism

Glyoxylate and dicarboxylate metabolismRiboflavin metabolism

Pentose phosphate pathwayD-Arginine and D-ornithine metabolism

alpha-Linolenic acid metabolismGlutathione metabolism

Sphingolipid metabolismArachidonic acid metabolism

Alanine, aspartate and glutamate metabolismCitrate cycle (TCA cycle)

Selenoamino acid metabolismTaurine and hypotaurine metabolism

Propanoate metabolismValine, leucine and isoleucine degradation

Glycerolipid metabolismGlycine, serine and threonine metabolism

Pyruvate metabolismSynthesis and degradation of ketone bodiesValine, leucine and isoleucine biosynthesis

Porphyrin and chlorophyll metabolismGlycolysis or Gluconeogenesis

Butanoate metabolism

Pregnant Wt vs Pregnant Mpc1

0.04 0.050.00 0.01 0.02 0.03

Synthesis and degradation of ketone bodiesPhenylalanine metabolism

Biotin metabolismValine, leucine and isoleucine degradation

D-Arginine and D-ornithine metabolismAmino sugar and nucleotide sugar metabolism

Purine metabolismPyrimidine metabolism

Sulfur metabolismAminoacyl-tRNA biosynthesis

Nicotinate and nicotinamide metabolismArginine and proline metabolism

Valine, leucine and isoleucine biosynthesisPhenylalanine, tyrosine and tryptophan biosynthesis

Fructose and mannose metabolismArachidonic acid metabolism

Galactose metabolismGlyoxylate and dicarboxylate metabolism

Biosynthesis of unsaturated fatty acidsTaurine and hypotaurine metabolism

Fatty acid metabolismPrimary bile acid biosynthesis

Alanine, aspartate and glutamate metabolismSphingolipid metabolism

Glycerophospholipid metabolismTerpenoid backbone biosynthesis

Pantothenate and CoA biosynthesisGlycosylphosphatidylinositol(GPI)-anchor biosynthesis

alpha-Linolenic acid metabolismButanoate metabolism


Steroid biosynthesisNitrogen metabolism

Linoleic acid metabolismCysteine and methionine metabolism

Histidine metabolismGlycine, serine and threonine metabolism

Glycerolipid metabolismFatty acid biosynthesis

Starch and sucrose metabolismbeta-Alanine metabolism

Lysine degradationTyrosine metabolism

Citrate cycle (TCA cycle)Fatty acid elongation in mitochondria

Thiamine metabolismSteroid hormone biosynthesisSelenoamino acid metabolism

Glutathione metabolismLysine biosynthesis


Pregnant Wt vs Pregnant DKO

0.00 0.01 0.02 0.03 0.04 0.05

Phenylalanine metabolismFatty acid metabolismGalactose metabolism

Glycerophospholipid metabolismSphingolipid metabolism

Histidine metabolismAlanine, aspartate and glutamate metabolism

Butanoate metabolismSynthesis and degradation of ketone bodies

Arginine and proline metabolismTryptophan metabolism

Arachidonic acid metabolismCitrate cycle (TCA cycle)

Pentose and glucuronate interconversionsFatty acid elongation in mitochondria

Starch and sucrose metabolismNicotinate and nicotinamide metabolism

D-Glutamine and D-glutamate metabolismPyrimidine metabolismRiboflavin metabolism

Aminoacyl-tRNA biosynthesisSelenoamino acid metabolism

Pyruvate metabolismBiosynthesis of unsaturated fatty acidsD-Arginine and D-ornithine metabolism

Glycolysis or GluconeogenesisGlycine, serine and threonine metabolism

Cysteine and methionine metabolismTyrosine metabolism

Fatty acid biosynthesisSteroid hormone biosynthesis


Phenylalanine, tyrosine and tryptophan biosynthesisPropanoate metabolism

Glycosylphosphatidylinositol(GPI)-anchor biosynthesisGlutathione metabolism

Steroid biosynthesisGlyoxylate and dicarboxylate metabolism

Glycerolipid metabolismPorphyrin and chlorophyll metabolism


Fructose and mannose metabolismValine, leucine and isoleucine biosynthesis

Linoleic acid metabolismalpha-Linolenic acid metabolism

Ubiquinone and other terpenoid-quinone biosynthesis

Pregnant Cpt2 vs Pregnant PPAR-α

0.00 0.02 0.04 0.06

Phenylalanine metabolismSphingolipid metabolism

Galactose metabolismStarch and sucrose metabolism

Synthesis and degradation of ketone bodiesButanoate metabolism

Citrate cycle (TCA cycle)Pyruvate metabolism

Glycolysis or GluconeogenesisGlycerophospholipid metabolism

Propanoate metabolismPentose and glucuronate interconversions

Pyrimidine metabolismalpha-Linolenic acid metabolism

Riboflavin metabolismAlanine, aspartate and glutamate metabolism

Selenoamino acid metabolismArachidonic acid metabolism

D-Arginine and D-ornithine metabolismSteroid biosynthesis

Glycosylphosphatidylinositol(GPI)-anchor biosynthesisSteroid hormone biosynthesis


Tryptophan metabolismAminoacyl-tRNA biosynthesis

Fructose and mannose metabolismArginine and proline metabolism

Histidine metabolismGlyoxylate and dicarboxylate metabolismGlycine, serine and threonine metabolism

Fatty acid metabolismPhenylalanine, tyrosine and tryptophan biosynthesis

Cysteine and methionine metabolismD-Glutamine and D-glutamate metabolism

Nicotinate and nicotinamide metabolismBiosynthesis of unsaturated fatty acids

Lysine biosynthesisGlycerolipid metabolism

Tyrosine metabolismBiotin metabolism

Glutathione metabolism

Pregnant DKO vs Pregnant PPAR-α

1.50.0 0.5 1.0Biotin metabolism

Amino sugar and nucleotide sugar metabolismPyrimidine metabolism

Starch and sucrose metabolismGlycosylphosphatidylinositol(GPI)-anchor biosynthesis

Primary bile acid biosynthesisArginine and proline metabolism

Lysine biosynthesisHistidine metabolism

Glyoxylate and dicarboxylate metabolismPurine metabolism

Taurine and hypotaurine metabolismNitrogen metabolism

Citrate cycle (TCA cycle)Lysine degradation

Riboflavin metabolismbeta-Alanine metabolism

Nicotinate and nicotinamide metabolismPropanoate metabolism

Aminoacyl-tRNA biosynthesisFructose and mannose metabolism

Alanine, aspartate and glutamate metabolismTyrosine metabolism

Synthesis and degradation of ketone bodiesGlutathione metabolism


Fatty acid metabolismGlycine, serine and threonine metabolism

Porphyrin and chlorophyll metabolismGlycerophospholipid metabolismalpha-Linolenic acid metabolism

Sulfur metabolismValine, leucine and isoleucine biosynthesisValine, leucine and isoleucine degradation

Fatty acid biosynthesisButanoate metabolismVitamin B6 metabolism

Fatty acid elongation in mitochondriaPyruvate metabolismThiamine metabolism

Biosynthesis of unsaturated fatty acidsSteroid biosynthesis

Tryptophan metabolismGlycolysis or Gluconeogenesis

Pentose phosphate pathwayMethane metabolism

Cyanoamino acid metabolismD-Arginine and D-ornithine metabolism

D-Glutamine and D-glutamate metabolismPantothenate and CoA biosynthesis

Pentose and glucuronate interconversionsUbiquinone and other terpenoid-quinone biosynthesis

Galactose metabolismSteroid hormone biosynthesis

Phenylalanine, tyrosine and tryptophan biosynthesisTerpenoid backbone biosynthesis

Glycerolipid metabolismLinoleic acid metabolism

Cysteine and methionine metabolismPhenylalanine metabolism

Selenoamino acid metabolismArachidonic acid metabolism

Sphingolipid metabolism

Pregnant Cpt2 vs Pregnant DKO

0.00 0.02 0.04 0.06

Starch and sucrose metabolismAmino sugar and nucleotide sugar metabolism

Phenylalanine metabolismGalactose metabolism

Pyrimidine metabolismAminoacyl-tRNA biosynthesis

Biotin metabolismArginine and proline metabolism

Glyoxylate and dicarboxylate metabolismGlycerophospholipid metabolism


Nicotinate and nicotinamide metabolismD-Arginine and D-ornithine metabolism

Arachidonic acid metabolismSphingolipid metabolism

Synthesis and degradation of ketone bodiesLysine degradation

Valine, leucine and isoleucine degradationFructose and mannose metabolism

Biosynthesis of unsaturated fatty acidsTerpenoid backbone biosynthesis

Fatty acid metabolismalpha-Linolenic acid metabolism

Histidine metabolismPantothenate and CoA biosynthesis

Steroid biosynthesisLinoleic acid metabolism

Glycine, serine and threonine metabolismPentose and glucuronate interconversionsValine, leucine and isoleucine biosynthesis

Fatty acid biosynthesisLysine biosynthesis

beta-Alanine metabolismNitrogen metabolism

Tryptophan metabolismButanoate metabolism

Taurine and hypotaurine metabolismFatty acid elongation in mitochondriaCysteine and methionine metabolism

Glycosylphosphatidylinositol(GPI)-anchor biosynthesisGlycerolipid metabolism

Purine metabolismPhenylalanine, tyrosine and tryptophan biosynthesis

Sulfur metabolismGlutathione metabolismPropanoate metabolism

Steroid hormone biosynthesisCitrate cycle (TCA cycle)

Riboflavin metabolismThiamine metabolism

0.01

0.02

0.03

0.04

Color Codes

p-value

p-value

0.01

0.02

0.03

0.04

0.4

0.6

0.8

0.01

0.02

0.03

0.04

0.00 0.02 0.04 0.06

Arginine and proline metabolismCysteine and methionine metabolism

Butanoate metabolismPhenylalanine metabolism

Galactose metabolismGlyoxylate and dicarboxylate metabolismGlycine, serine and threonine metabolism


Starch and sucrose metabolismTryptophan metabolism

Aminoacyl-tRNA biosynthesisHistidine metabolism

alpha-Linolenic acid metabolismPentose and glucuronate interconversions

Sphingolipid metabolismCitrate cycle (TCA cycle)

Alanine, aspartate and glutamate metabolismPropanoate metabolismFatty acid biosynthesis

Glycerophospholipid metabolismFructose and mannose metabolism

Amino sugar and nucleotide sugar metabolismD-Glutamine and D-glutamate metabolism

Lysine degradationNicotinate and nicotinamide metabolism

Pyrimidine metabolismLysine biosynthesis

Linoleic acid metabolismSynthesis and degradation of ketone bodies

D-Arginine and D-ornithine metabolismValine, leucine and isoleucine degradation

Purine metabolismNitrogen metabolism

Fatty acid elongation in mitochondriaMethane metabolism

Cyanoamino acid metabolismPyruvate metabolism

Pantothenate and CoA biosynthesisBiosynthesis of unsaturated fatty acids

Tyrosine metabolismFatty acid metabolismRiboflavin metabolism

Valine, leucine and isoleucine biosynthesisSelenoamino acid metabolism

Glycerolipid metabolismGlycolysis or Gluconeogenesis

Phenylalanine, tyrosine and tryptophan biosynthesisSulfur metabolism

Vitamin B6 metabolismbeta-Alanine metabolism

Pregnant Mpc1 vs Pregnant PPAR-α

0.01

0.02

0.03

0.04

0.00 0.01 0.02 0.03 0.04 0.05

Fatty acid metabolismBiosynthesis of unsaturated fatty acids

Nicotinate and nicotinamide metabolismFatty acid elongation in mitochondria

Pyrimidine metabolismFatty acid biosynthesis

Phenylalanine metabolismGalactose metabolism

Arginine and proline metabolismArachidonic acid metabolism

D-Arginine and D-ornithine metabolismAmino sugar and nucleotide sugar metabolism

Pantothenate and CoA biosynthesisLinoleic acid metabolism

Starch and sucrose metabolismAminoacyl-tRNA biosynthesis

alpha-Linolenic acid metabolismHistidine metabolism

beta-Alanine metabolismTerpenoid backbone biosynthesis

Synthesis and degradation of ketone bodiesGlycine, serine and threonine metabolism

Phenylalanine, tyrosine and tryptophan biosynthesisFructose and mannose metabolism

Lysine degradationSphingolipid metabolism


Cysteine and methionine metabolismTryptophan metabolism

Glyoxylate and dicarboxylate metabolismValine, leucine and isoleucine degradation

Nitrogen metabolismSteroid biosynthesis

Glycerophospholipid metabolismValine, leucine and isoleucine biosynthesis

Biotin metabolismTaurine and hypotaurine metabolism

Glutathione metabolismSteroid hormone biosynthesis

Purine metabolismSulfur metabolism

Primary bile acid biosynthesisGlycosylphosphatidylinositol(GPI)-anchor biosynthesis

Pentose and glucuronate interconversionsLysine biosynthesis

Butanoate metabolismGlycerolipid metabolism

Pregnant Mpc1 vs Pregnant Cpt2

0.01

0.02

0.03

0.04

0.01

0.02

0.03

0.01

0.02

0.03

0.04

0.6

0.8

Pregnant Mpc1 vs Pregnant DKO

0.01

0.02

0.03

0.04

0.00 0.02 0.04 0.06

Glycine, serine and threonine metabolismCysteine and methionine metabolism

Arginine and proline metabolismSynthesis and degradation of ketone bodies

Butanoate metabolismGalactose metabolism

Glyoxylate and dicarboxylate metabolismTryptophan metabolism

Citrate cycle (TCA cycle)Aminoacyl-tRNA biosynthesis


Histidine metabolismPentose and glucuronate interconversions

Purine metabolismAlanine, aspartate and glutamate metabolism

alpha-Linolenic acid metabolismPhenylalanine metabolism

Valine, leucine and isoleucine degradationSulfur metabolism

Pyruvate metabolismStarch and sucrose metabolism

Fatty acid biosynthesisSphingolipid metabolism

Valine, leucine and isoleucine biosynthesisNicotinate and nicotinamide metabolism

D-Arginine and D-ornithine metabolismD-Glutamine and D-glutamate metabolism

Lysine degradationGlycerophospholipid metabolism


Riboflavin metabolismFructose and mannose metabolism

Pyrimidine metabolismTyrosine metabolism

Glycerolipid metabolismLysine biosynthesis

Glycolysis or GluconeogenesisLinoleic acid metabolism

Selenoamino acid metabolismFatty acid metabolism

Biotin metabolismAmino sugar and nucleotide sugar metabolism

Propanoate metabolismBiosynthesis of unsaturated fatty acids

Pantothenate and CoA biosynthesisNitrogen metabolism

Thiamine metabolismFatty acid elongation in mitochondria

Phenylalanine, tyrosine and tryptophan biosynthesisGlutathione metabolism

Taurine and hypotaurine metabolism

Pregnant Wt vs Pregnant PPAR-α

0.02

0.04

0.00 0.01 0.02 0.03 0.04

Aminoacyl-tRNA biosynthesisGlycosylphosphatidylinositol(GPI)-anchor biosynthesis

Histidine metabolismMethane metabolism

Cyanoamino acid metabolismPantothenate and CoA biosynthesis

Steroid hormone biosynthesisVitamin B6 metabolism

Biotin metabolismGlycine, serine and threonine metabolism

Glycerophospholipid metabolismLysine degradation

Amino sugar and nucleotide sugar metabolismPyrimidine metabolism

Glutathione metabolismNicotinate and nicotinamide metabolism

Phenylalanine, tyrosine and tryptophan biosynthesisArginine and proline metabolism

Phenylalanine metabolismArachidonic acid metabolism

Sphingolipid metabolismCysteine and methionine metabolism


Starch and sucrose metabolismCitrate cycle (TCA cycle)

Glyoxylate and dicarboxylate metabolismTryptophan metabolism

alpha-Linolenic acid metabolismPentose and glucuronate interconversions

Nitrogen metabolismPorphyrin and chlorophyll metabolism

Terpenoid backbone biosynthesisValine, leucine and isoleucine degradation

Primary bile acid biosynthesisButanoate metabolismGalactose metabolism

Linoleic acid metabolismPyruvate metabolismTyrosine metabolism

Non-Pregnant Wt vs Pregnant Wt

0.01

0.02

0.03

Figure S2

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

p-value

Color Codesp-value

p-value

Color Codes

p-value

p-value

Color Codes

p-value

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Maternal Lipid Metabolism Directs Fetal Liver Programming … · 2019-10-30 · Fasting induces...

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